Our hypothesis is that TREX1 and TREX2 are exonucleases with 3'-[unreadable]5' excision activity that is important for removing problematic nucleotides during either DNA replication and/or DMArepair and our goal is to study these proteins in cells and mice. Currently TREX1 and TREX2 are thought to be exonucleases based on their homology to three exonuclease sequence motifs of E. coli DNA pol 1 large fragment and bacteriophage T4 DNA pol and based on their 3'-[unreadable]5' exonuclease activity in vitro. However, at this time there is no more published information defining their cellular function. Thus, TREX1 and TREX2 are potentially important for maintaining genomic stability and perhaps cancer prevention. There are three aims that define TREX1 and TREX2. Aim 1: to elucidate fundamental TREX1 and TREX2 biochemical functions. There three known biochemical functions: 1) self association 2) exonuclease activity, 3) and DNA binding activity. We have setup in vitro assays to observe these activities and our goal is to generate impaired forms of TREX1 and TREX2 that perform some but not all of these activities. Aim 2: to discover the biological importance of TREX1 and TREX2 by analyzing genetically altered mouse embryonic stem (ES) cells. We will compare a mutation that is null to mutations that alter some but not all functions as discovered in aim 1. At this time we have generated TREX2-null ES cells and our preliminary results demonstrate that TREX2 is indeed involved in genome maintenance. We show TREX2-null cells exhibit altered sensitivity to some DNA damaging agents and TREX2-null cells exhibit genomic instability including gross chromosomal rearrangements. Aim 3: to analyze TREX-null mice in wild type and p53 mutant backgrounds. Wewill perform a life span analysis and determine the onset, incidence and spectra of age-related characteristics including cancer and genomic instability. The impact p53-mediated responses to damaged DNA will be determined by studying double-mutant mice. Completion of this proposal will greatly facilitate our understanding of TREX1 and TREX2 for maintaining genome stability and for preventingcancer.